Our long-term goal is to elucidate the mechanism of biofilm tolerance to antibiotics. Our preliminary studies suggest that persister cells may be largely responsible for resistance of biofilms and stationary planktonic populations to killing by cidal antimicrobials. The main goal of this proposal is to identify genes responsible for the persister phenotype. This will enable us to directly test the persister hypothesis of biofilm resistance which promises to solve this long-standing riddle, and will provide a new paradigm for the understanding and treatment of biofilm infections. We will use a number of complementary approaches to identify persister genes. We were able to isolate persisters from a high-persistence (hip) strain of E. coli by lysing the bulk of cells with ampicillin, and obtained a preliminary gene expression profile. A detailed time-dependent gene profile of ampicillin treatment will be obtained, providing data for a comprehensive cluster analysis that will indicate candidate persister genes. We will isolate naive persisters using cell sorting with GFP linked to genes that are likely to be expressed in these cells. Additionally, using DNA arrays, we will identify an overlapping set of genes differentially expressed in cells treated with unrelated antibiotics. Persister genes are expected to be among those affecting death and survival. In an independent approach, persister genes will be identified by selection for increased tolerance from a recombinant genomic library. Candidate genes from these approaches will be tested in uniformly constructed strains, each carrying a deletion; and overexpressing the gene from a controllable promoter. Tests with a set of antibiotics will indicate genes that affect persister production in planktonic cultures. Biofilms will then be prepared from persister-deficient or overproducing strains, and tested for tolerance with cidal antibiotics. Correlation between persister status of a strain and biofilm tolerance will provide a definitive test for the persister hypothesis. Identified persister genes will then enable a study of their mechanism of action, which will begin with obtaining an expression profile from strains deficient in; and overproducing the protein of interest. These studies will form the basis for understanding biofilm infections and developing drugs that target persister proteins.